1. Field of the Invention
The present invention relates to uniformly-sized, small microspheres, to methods of making the microspheres and to their use in labeling biological cell surfaces.
2. Description of the Prior Art
The isolation and characterization of cell membranes and their components is essential for an understanding of the role in which surface membranes play in regulating a wide variety of biological and immunological activities. The present techniques used for this purpose are not quite satisfactory.
Knowledge of the nature, number and distribution of specific receptors on cell surfaces is of central importance for an understanding of the molecular basis underlying such biological phenomena as cell-cell recognition in development, cell communication and regulation by hormones and chemical transmitters, and differences in normal and tumor cell surfaces. In previous studies, the localization of antigens and carbohydrate residues on the surface of cells, notably red blood cells and lymphocytes, has been determined by bonding antibodies or lectins to such macromolecules as ferritin, hemocyanin or peroxidase which have served as markers for transmission electron microscopy. With advances in high resolution scanning electron microscopy (SEM), however, the topographical distribution of molecular receptors on the surfaces of cell and tissue specimens can be readily determined by similar histochemical techniques using newly developed markers resolvable by SEM.
Recently commercially available polystyrene latex particles have been utilized as immunologic markers for use in the SEM technique. The surface of such polystyrene particles is hydrophobic and hence certain types of macromolecules such as antibodies are absorbed on the surface under carefully controlled conditions. However, such particles stick non-specifically to many surfaces and molecules and this seriously limits their broad application.
The preparation of small, stable spherical particles which are bio-compatible, i.e., do not interact non-specifically with cells or other biological components and which contain functional groups to which specific proteins and other bio-chemical molecules can be covalently bonded is disclosed in copending application Ser. No. 434,124, filed Jan. 17, 1974, now issued on May 18, 1976, as U.S. Pat. No. 3,957,741.
Smaller, more evenly shaped microspheres are disclosed in Ser. No. 634,935, filed Nov. 24, 1975, now issued on Feb. 6, 1979, as U.S. Pat. No. 4,138,383 and microspheres having a density differing from that of cell membranes are disclosed in Ser. No. 634,929, filed Nov. 24, 1975, now issued on July 12, 1977, as U.S. Pat. No. 4,035,316.
The hydroxyl groups can be activated by cyanogen bromide for covalent bonding of proteins and other chemicals containing amino groups to the polymericlatex. Methacrylic acid residues which impart a negative charge onto the particles are likely to prevent non-specific binding to cell surfaces and to provide carboxyl groups to which a variety of bio-chemical molecules can be covalently bonded using the carbodiimide method. Cross-linking of the polymeric matrix is preferable in order to maintain the stability and size of the particles in both aqueous solution and in organic solvents commonly used in the fixation and dehydration of biological specimens for electron or light microscopy.
Microspheres, 150-350 A in diameter serve as markers for transmission electron microscopy as well as in high resolution scanning electron microscopy. Microspheres larger than 0.2 micron in diameter can be utilized with ordinary visual microscopy. However, the attachment of fluorescent tags to the surface required a covalent bonding reaction and the distribution of tags was not totally uniform and the attachment resulted in a consumption of covalent bonding sites which would otherwise be available for marking with antigen, lectin or antibody.